Objective To investigate the role of β-catenin in the differentiation of C3H10T1/2 cells into cardiomyocyte-like cells induced by bone morphogenetic protein 9 (BMP9). Methods C3H10T1/2 cells were transfected with the recombinant adenovirus expressing BMP9 (Ad-BMP9) and differentiated into cardiomyocytes in vitro for up to 21 days. The activated level of β-catenin in the cells after cultivated with Ad-BMP9 and different concentrations of β-catenin specific inhibitor XAV-939 was detected by Western blotting. Real-time quantitative PCR (qRT-PCR) was performed to evaluate the expressions of cardiac specific gene myocyte enhancer factor 2C (MEF2C) and GATA binding protein 4 (GATA4) in the cells one week after induced by Ad-BMP9 and different concentrations of XAV-939. Three weeks after the transfection, the expressions of connexin 43 (Cx43) and cardiac troponin T (cTnT) were analyzed by Western blotting, and the location of Cx43 in the cells was observed by immunofluorescence technique. Results While the transfection was successful in 50% of the cells positive for BMP9, β-catenin was excessively activated and their phosphorylation level remarkably increased. After XAV-939 inhibited the activity of β-catenin, the expressions of MEF2C, GATA4, Cx43, cTnT in C3H10T1/2 cells induced by BMP9 were significantly suppressed. Conclusion The β-catenin can be activated by BMP9 and the activation of β-catenin plays an important role in the differentiation of C3H10T1/2 cells into cardiomyocyte-like cells induced by BMP9.

Bone morphogenetic protein (BMP) 9 (BMP9) is one of most potent BMPs in inducing osteogenic differentiation of mesenchymal stem cells (MSCs). Recently, evidence has shown that osteogenesis and angiogenesis are coupled, however, it is unclear whether BMP9 induces MSC differentiation into endothelial-like cells and further promotes blood vessel formation. In the present study, we explored the potential of BMP9-induced angiogenic differentiation of MSCs, and the relationship between BMP9-induced osteogenic and angiogenic differentiation of MSCs. Osteogenic activities and angiogenic differentiation markers were analyzed at mRNA and protein levels. In vivo osteogenic and angiogenic differentiation of MSCs were tested by the ectopic bone formation model. We identified that adenoviral vectors effectively transduced in immortalized mouse embryonic fibroblasts (iMEFs) and expressed BMP9 with high efficiency. We found that BMP9 induces early and late osteogenic differentiation, and it up-regulated osteogenic marker expression in MSCs. Meanwhile, BMP9 induces angiogenic differentiation of MSCs via the expression of vascular endothelial growth factor a (VEGFa) and CD31 at both mRNA and protein levels. CD31-positive cells were also increased with the stimulation of BMP9. The ectopic bone formation tests found that BMP9-induced trabecular bone formation was coupled with the expression of blood vessel formation markers and sinusoid capillary formation. These findings suggest that BMP9 exhibits dual and coupled roles in inducing osteogenic and angiogenic differentiation of MSCs.

Keywords: Angiogenic differentiation; BMP9; MSCs; Osteogenesis-angiogenesis coupling; Osteogenic differentiation.

OBJECTIVE

To investigate the roles of extracellular signal-regulated kinase 5 (ERK5) and c-Jun N-terminal kinase (JNK) in the differentiation of C3H10T1/2 cells into cardiomyocyte-like cells induced by bone morphogenetic protein 9 (BMP9).

METHODS

BMP9 gene was imported into C3H10T1/2 cells by recombinant adenovirus. Western blotting was used to detect the actived levels of ERK5 and JNK after cultivated with BMP9 and different concentrations of ERK5 specific inhibitor BIX02189 or JNK specific inhibitor SP600125; real-time quantitative PCR (qRT-PCR) was performed to analyze the expressions of myocardium specific genes GATA binding protein 4 (GATA4), myocyte enhancer factor 2C (MEF2C) after one week induced by BMP9; Western blotting was conducted to measure the expressions of myocardium specific proteins connexin 43 (CX43), cardiac troponin T (cTnT) after three weeks induced by BMP9, and immunofluorescence to observe the positions of CX43 and cTnT in the cells.

RESULTS

In the case of transfection efficiency up to 50%, BMP9 exceedingly activated ERK5 and JNK, and significantly increased their phosphorylation level (P<0.05). After BIX02189 inhibited the activity of ERK5, the expression levels of myocardial differentiation markers MEF2C, GATA4, CX43, cTnT of C3H10T1/2 cells were significantly suppressed (P<0.05); JNK specific inhibitor SP600125 also inhibited the expression levels of MEF2C, GATA4, CX43, cTnT, but the inhibition on MEF2C and GATA4 was not as notable as that of BIX02189 (P<0.05).

CONCLUSIONS

The excessive activation of ERK5 and JNK plays an important role in the differentiation of C3H10T1/2 cells into cardiomyocyte-like cells induced by BMP9.

The late vitellogenic stage of the mud crab is characterized by large and obvious follicle cells as well as an enlarged oocyte nucleus and a prominent germinal vesicle (GV). The aim of this study was evaluation of functions of oocytes and follicle cells during meiosis as well as at identifying associated ovarian autocrine/paracrine factors using comparative transcriptomics. The results from the KEGG pathway analysis indicated DNA replication, nucleotide excision repair, spliceosome and the ribosome pathways were highly associated with oocyte maturation across both transcriptomes. In addition, there was a larger abundance of mRNA transcripts for cell cycle-related genes in the oocyte, as well as cyclin A, cyclin B and CKS1B in the GV than at the time of germinal vesicle breakdown (GVBD). These findings indicate these cell cycle-related genes might be involved in GVBD induction. Results when there was localization of ligands and the respective receptors of VEGF, TGFβ propeptide and BMP9/10 indicated these proteins might be autocrine/paracrine factors. Results from functional analysis of VEGF, TGFβ propeptide and BMP9/10 in oocyte maturation using RNA interference revealed that these proteins might be involved in oocyte maturation by regulating cyclin abundance. This is the first study on the functions of VEGF in oocyte maturation in invertebrates.

Keywords: Autocrine/paracrine factors; Cyclin; Illumina sequencing; Oocyte maturation; Scylla paramamosain.

Intervertebral disk degeneration (IDD) is a common disease that is caused by degeneration of the nucleus pulposus (NP). One goal in the treatment of IDD is delaying or reversing the degeneration of NP via the transformation of exogenous genes. This study first investigated the role of BMP9 in the extracellular matrix (ECM) of nucleus pulposus cells (NPCs) and its mechanism. We found that BMP9 promotes the expression of ECM in NPCs, and the key molecules of Notch signaling, namely, NICD-1, hes and hey, and it was significantly altered in BMP9-transfected NPCs, which suggests that BMP9 may regulate the ECM via the Notch signaling pathway. We verified the expression of Notch ligands and receptors in NPCs infected with Ad-BMP9 and demonstrated a significant decrease in DLL1 and Notch1; then, NPCs were transfected with Ad-dnNotch1, Ad-Jagged1, and Ad-DLL1, and different multiple groups were established to further identify the ligands or receptors that affected ECM expression. The results demonstrated that Ad-dnNotch1, Jagged1 and DLL1 inhibited ECM expression, and dnNotch1 promoted expression. Therefore, we demonstrated that BMP9 promoted the expression of ECM in NPCs via inhibition of Notch1 and DLL1. This study provides a possible method for IDD treatment.

OBJECTIVE

To compare the serum proangiogenic biomarkers in diabetic patients suffering from with and without diabetic retinopathy (DR).

METHODS

An observational study.

METHODS

Arif Memorial Teaching Hospital, Lahore, Pakistan and Institute of Molecular Biology and Biotechnology/Centre for Research in Molecular Medicine, The University of Lahore, Lahore, Pakistan, from March to December 2017.

METHODS

Forty patients with DR were included in group A and 15 patients without retinopathy (controls) were included in group B. Twelve serum pro-angiogenic biomarkers [Angiopoietin 2, Human Growth Factor (HGF), Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), Placental Growth Factor (PLGF), Vascular Endothelial Growth Factor-A and C (VEGF-A and VEGF-C), Bone Morphogenetic Protein 9 (BMP9), Follistatin, Leptin, Interleukin-8 (IL8), Endothelin (ET)] were analysed by xMAP flow cytometry technique, results were compared between the two groups and statistical analysis was done using Mann-Whitney U test.

RESULTS

Serum ET, Follistatin and EGF were significantly raised in group A as compared to group B having p-values of 0.001, <0.001, and 0.033, respectively. Serum BMP9, Leptin, HGF, FGF and VEGF-C had p <0.001, 0.023, 0.020, and 0.009, respectively and were higher in group B than group A.

CONCLUSIONS

Serum ET, Follistatin and EGF were significantly higher in DR patients as compared to those without DR and should be considered to be significant biomarkers of retinal complications in diabetes mellitus.

Fibrosis is a common endpoint for chronic organic diseases. Many signaling pathways and cytokines are involved in the development of fibrosis. Recently, bone morphogenetic protein 9 (BMP9), a member of transforming growth factor-beta (TGF-β) superfamily, has been considered as a pivotal regulator in tissue fibrosis, and exerts its diverse effects on the activation of fibroblasts and the development of fibrotic diseases. BMP9 exhibits its functional roles largely through binding to type I BMP receptor activin receptor-like kinase 1 (ALK1), and then directly activates small mother against decapentaplegic (Smad) signaling which promotes or limits the expressions of pro-fibrotic genes. Accumulating studies have demonstrated that the aberrant BMP9/ALK1/Smad signaling pathway affects the fibrogenesis of various organs, but the exact role of BMP9 in fibrosis remains elusive and debatable. In the present review, reports from in vitro, in vivo and clinical studies regarding the functions and underlying mechanisms of the BMP9 signaling in tissue fibrosis, are widely summarized and discussed. In addition, the major components of the BMP9 signaling pathway and the potential interventions targeting this pathway are also described. This review will enrich our understanding of the BMP9 signaling pathway in organ fibrosis, and provide a novel clue for the potential interventions of organ fibrosis.

Keywords: Bone morphogenetic protein 9; Fibroblast; Organ fibrosis; Signaling pathway.

Osteosarcoma (OS) causes millions of death worldwide and, since there is no effective therapy, it is necessary to identify the molecular mechanism of OS, which can direct the development of new therapies. This study investigated the role of bone morphogenetic protein 9 (BMP9), a member of the transforming growth factor (TGF)-β family, in OS development. This study first examined BMP9 expression in tissue from OS patients and normal subjects. The OS cell line (MG63) and tumor cells from OS patients were then transfected with BMP9 and cell proliferation and apoptosis were assessed. Western blot and reverse transcription-polymerase chain reaction were used to study the expression of cancer-related genes [B cell lymphoma (Bcl)-2, cleaved Caspase-3, Caspase-9, and poly ADP-ribose polymerase]. To confirm the in vivo impact of BMP9, mice were transplanted with OS tumor cells and then treated with BMP9 carried in attenuated Salmonella enterica serovar Typhimurium. Our study found that the OS tumor tissue had a lower expression of BMP9 compared to normal tissue. Transfection of BMP9 in OS and MG63 cells inhibited cell growth and promoted apoptosis. In vitro studies showed a decrease in Bcl-2 gene expression and an increase in Cyto-c, Caspase-3, and Caspase-9 expression. In vivo studies indicated that consistent treatment with BMP9 in OS mice results in inhibition of tumor growth. This study shows that BMP9 inhibition is associated with OS development and that enhanced expression of BMP9 may be a potential treatment method for OS.

Characteristic bone metabolism was observed in obesity and diabetes with controversial conclusions. Type 2 diabetes (T2DM) and obesity may manifest increased bone mineral density. Also, obesity is more easily to occur in T2DM. Therefore, we infer that some factors may be linked to bone and obesity as well as glucose metabolism, which regulate all of them. Bone morphogenetic proteins (BMPs), belonging to the transforming growth factor- (TGF-) beta superfamily, regulate a diverse array of cellular functions during development and in the adult. More and more studies revealed that there exists a relationship between bone metabolism and obesity as well as glucose metabolism. BMP2, BMP4, BMP6, BMP7, and BMP9 have been shown to affect the pathophysiological process of obesity and glucose metabolism beyond bone metabolism. They may exert functions in adipogenesis and differentiation as well as insulin resistance. In the review, we summarize the literature on these BMPs and their association with metabolic diseases including obesity and diabetes.

Pulmonary veno-occlusive disease (PVOD) occurs in humans either as heritable form (hPVOD) due to biallelic inactivating mutations of EIF2AK4 (encoding GCN2), or as a sporadic form at older age (sPVOD). The chemotherapeutic agent Mitomycin C is a potent inducer of PVOD in humans and in rats (MMC-PVOD). Here we compared human hPVOD and sPVOD, and MMC-PVOD pathophysiology at the histological, cellular and molecular levels to unravel common altered pathomechanisms. MMC-exposure in rats was primarily associated with arterial and microvessels remodeling and secondarily followed by venous remodeling, when PVOD became symptomatic. In all forms of PVOD tested, there were convergent GCN2-dependent but eIF2α-independent pulmonary protein overexpression of heme oxygenase 1 (HO-1) and CCAAT-enhancer-binding protein (C/EBP) homologous protein (CHOP), two downstream effectors of GCN2 signaling and endoplasmic reticulum (ER) stress. In human PVOD samples, CHOP immunohistochemical staining mainly labeled endothelial cells in remodeled veins and arteries. Strong HO-1 staining was observed only within capillary hemangiomatosis foci, where intense microvascular proliferation occurs. HO-1 and CHOP stainings were not observed in control and pulmonary arterial hypertension lung tissues, supporting the specificity for CHOP and HO-1 involvement in PVOD pathobiology. In vivo loss of GCN2 (EIF2AK4 mutations carriers and Eif2ak4-/- rats) or in vitro GCN2 inhibition in cultured pulmonary artery endothelial cells (PAECs) using pharmacological and siRNA approaches demonstrated that GCN2 loss-of-function negatively regulates BMP-dependent SMAD1/5/9 signaling. Exogenous BMP9 was still able to reverse GCN2 inhibition-induced PAECs proliferation. In conclusion, we identified CHOP and HO-1 inhibition, and BMP9 as potential therapeutic options for PVOD.

OBJECTIVE

We previously reported that low intensity pulsed ultrasound (LIPUS) promotes marrow stromal cell (MSC) osteogenesis and suppresses the LPS-induced inflammatory response in osteoblasts. Here, we examined the effects of LIPUS on human periodontal ligament-derived stem cells (hPDLSCs) in chronic inflammatory bone disease, such as periodontitis.

METHODS

hPDLSCs were collected from 3 healthy third molars. hPDLSCs were induced to differentiate by either recombinant BMP2 or BMP9 with or without daily LIPUS treatment (20 min/d). hPDLSCs were also stimulated by Porphyromonas gingivalis-derived LPS (LPS-PG), IL-1beta, and TNF-alpha with or without LIPUS. Matrix mineralization was evaluated by alizarin red S staining. The expression of genes for osteogenic makers and for inflammatory cytokines were analyzed by real time RT-PCR.

RESULTS

LIPUS promoted BMP9-induced osteogenesis of hPDLSCs based on increases in both cell calcification and osteogenic marker expression. In contrast, LIPUS did not affect BMP2-induced osteogenic differentiation. LIPUS-induced Noggin expression was potentially involved in the differential response of the cells. Either LPS-PG, IL-1beta, or TNF-alpha-induced ERK phosphorylation and IL-8, CCL2, and RANKL expression were decreased in LIPUS-treated hPDLSCs. Moreover, the inhibitory effects of LPS-PG and IL-1beta on osteogenesis of hPDLSCs were significantly blocked by LIPUS.

CONCLUSIONS

LIPUS is an effective tool to promote osteogenic differentiation under inflammatory conditions.

This study investigated the role of muscle damage in bone defect healing using skull and tibial double-defect and tibial fracture models in dystrophin^-/-/Utrophin^-/- double-knockout (dKO-Hom) mice. The skull and tibia bone defect and fracture healing was monitored using micro-CT, histology, immuohistochemistry and quantitative PCR. We found the skull defect healing is not impaired while the tibial defect healing was delayed at day 7 in the dKO-Hom group compared to wild-type (WT) group as revealed by micro-CT. Mechanistically, the number of osteoclasts and osteoblasts significantly decreased in the defect area in dKO-Hom group compared to WT group on day 21. DKO-Hom mice showed higher mortality after fracture (6/12) and significantly impaired fracture healing compared to the other groups as revealed by the micro-CT parameters of the calluses. Histology showed higher osteoclast number in the calluses of dKO-Hom mice than other groups. Furthermore, dKO-Hom mice showed down-regulation of 15-Pgdh, Il-4, Bmp7, and Bmp9 at 10 days after tibia fracture and BMP6 and 7 in the muscle. In conclusion, the long bone defect and fracture healing are impaired in dKO-Hom mice which demonstrated significantly muscle sarcopenia and related with disturbance of osteoclastogenesis and osteoblastogenesis. The impaired tibial fracture healing was associated with down-regulation of several genes in the muscle.

Keywords: Muscular dystrophy; bone defect healing; dystrophin/utrophin double knockout; tibia fracture.

Bone morphogenetic protein (BMP) 9 is one of the most osteogenic BMPs, but its mechanism of action has not been fully elucidated. Hes1, a transcriptional regulator with a basic helix-loop-helix (bHLH) domain, is a well-known effector of Notch signaling. Here, we find that BMP9 induces periodic increases of Hes1 mRNA and protein expression in osteoblasts, presumably through an autocrine negative feedback mechanism. BMP9-mediated Hes1 induction is significantly inhibited by an ALK inhibitor and overexpression of Smad7, an inhibitory Smad. Luciferase and chromatin immunoprecipitation assays revealed that two Smad binding sites in the 5' upstream region of the mouse Hes1 gene are essential for transcriptional activation by BMP9. Thus, our data indicate that BMP9 induces Hes1 expression in osteoblasts via the Smad signaling pathway.

Keywords: Bone Morphogenetic Protein 9 (BMP9); Bone regeneration; Hes1; Notch; Osteogenic differentiation.

We synthesized 1 (San78-130), a reversible version of L-783277, as a selective and potent ALK1 inhibitor. Our study showed that 1 possesses great kinase selectivity against a panel of 342 kinases and more potent activity against ALK1 than L-783277. Among the six ALK isotypes (ALK1-6), ALK1 is most significantly inhibited by compound 1. Compound 1 suppresses the BMP9-induced Smad1/5 pathway by mainly inhibiting ALK1 in C2C12 cells. Our molecular dynamics simulations suggest that H-bonding interaction between the C-4' hydroxyl group of 1 and Arg334 of ALK1 substantially contributes to the ALK1 inhibition. To the best of our knowledge, 1 is the first selective ALK1 inhibitor. Furthermore, compound 1 promoted angiogenesis in both endothelial tube formation and microfluidic chip based 3D angiogenesis assays, suggesting that 1 could be a lead compound for therapeutic angiogenesis agents. Our study may provide an insight into designing selective and potent inhibitors against ALK1.

Background: Glucocorticoid-induced osteoporosis (GIOP) is the most common secondary osteoporosis. Patients with GIOP are susceptible to fractures and the subsequent delayed bone union or nonunion. Thus, effective drugs and targets need to be explored. In this regard, the present study aims to reveal the possible mechanism of the anti-GIOP effect of all-trans retinoic acid (ATRA).

Methods: Bone morphogenetic protein 9 (BMP9)-transfected mesenchymal stem cells (MSCs) were used as an in vitro osteogenic model to deduce the relationship between ATRA and dexamethasone (DEX). The osteogenic markers runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and osteopontin were detected using real-time quantitative polymerase chain reaction, Western blot, and immunofluorescent staining assay. ALP activities and matrix mineralization were evaluated using ALP staining and Alizarin Red S staining assay, respectively. The novel genes associated with ATRA and DEX were detected using RNA sequencing (RNA-seq). The binding of the protein-DNA complex was validated using chromatin immunoprecipitation (ChIP) assay. Rat GIOP models were constructed using intraperitoneal injection of dexamethasone at a dose of 1 mg/kg, while ATRA intragastric administration was applied to prevent and treat GIOP. These effects were evaluated based on the serum detection of the osteogenic markers osteocalcin and tartrate-resistant acid phosphatase 5b, histological staining, and micro-computed tomography analysis.

Results: ATRA enhanced BMP9-induced ALP, RUNX2 expressions, ALP activities, and matrix mineralization in mouse embryonic fibroblasts as well as C3H10T1/2 and C2C12 cells, while a high concentration of DEX attenuated these markers. When DEX was combined with ATRA, the latter reversed DEX-inhibited ALP activities and osteogenic markers. In vivo analysis showed that ATRA reversed DEX-inhibited bone volume, bone trabecular number, and thickness. During the reversal process of ATRA, the expression of retinoic acid receptor beta (RARβ) was elevated. RARβ inhibitor Le135 partly blocked the reversal effect of ATRA. Meanwhile, RNA-seq demonstrated that serine protease inhibitor, clade A, member 3N (Serpina3n) was remarkably upregulated by DEX but downregulated when combined with ATRA. Overexpression of Serpina3n attenuated ATRA-promoted osteogenic differentiation, whereas knockdown of Serpina3n blocked DEX-inhibited osteogenic differentiation. Furthermore, ChIP assay revealed that RARβ can regulate the expression of Serpina3n.

Conclusion: ATRA can reverse DEX-inhibited osteogenic differentiation both in vitro and in vivo, which may be closely related to the downregulation of DEX-promoted Serpina3n. Hence, ATRA may be viewed as a novel therapeutic agent, and Serpina3n may act as a new target for GIOP.

Keywords: ATRA; BMP9; Dexamethasone; Glucocorticoid-induced osteoporosis (GIOP); Osteogenic differentiation; Serpina3n.

Bone morphogenetic protein 9 (BMP9), also known as growth differentiation factor 2 (GDF2), is a member of the transforming growth factor β (TGF β) superfamily. Although BMP9 plays pivotal roles during development, including angiogenesis, hematopoiesis, hepatogenesis, osteogenesis, and glucose metabolism, little information is available for BMP9 expression in the central nervous system (CNS). We, thus, investigated BMP9 expression in the adult rat CNS using immunohistochemistry. BMP9 was intensely expressed in most neurons and their axons. Furthermore, we found that oligodendrocytes and ependymal cells also express BMP9 protein. These data indicate that BMP9 is widely expressed throughout the adult CNS, and this abundant expression strongly supports the idea that BMP9 also plays important roles in the adult brain.

Keywords: BMP9; GDF2; axon; immunohistochemistry; rat brain.

Teeth arise from the tooth germ through sequential and reciprocal interactions between immature epithelium and mesenchyme during development. However, the detailed mechanism underlying tooth development from tooth germ mesenchymal cells (TGMCs) remains to be fully understood. Here, we investigate the role of Wnt/β-catenin signalling in BMP9-induced osteogenic/odontogenic differentiation of TGMCs. We first established the reversibly immortalized TGMCs (iTGMCs) derived from young mouse mandibular molar tooth germs using a retroviral vector expressing SV40 T antigen flanked with the FRT sites. We demonstrated that BMP9 effectively induced expression of osteogenic markers alkaline phosphatase, collagen A1 and osteocalcin in iTGMCs, as well as in vitro matrix mineralization, which could be remarkably blunted by knocking down β-catenin expression. In vivo implantation assay revealed that while BMP9-stimulated iTGMCs induced robust formation of ectopic bone, knocking down β-catenin expression in iTGMCs remarkably diminished BMP9-initiated osteogenic/odontogenic differentiation potential of these cells. Taken together, these discoveries strongly demonstrate that reversibly immortalized iTGMCs retained osteogenic/odontogenic ability upon BMP9 stimulation, but this process required the participation of canonical Wnt signalling both in vitro and in vivo. Therefore, BMP9 has a potential to be applied as an efficacious bio-factor in osteo/odontogenic regeneration and tooth engineering. Furthermore, the iTGMCs may serve as an important resource for translational studies in tooth tissue engineering.

Keywords: BMP9; canonical Wnt/β-catenin signalling; osteo/odontogenesis; tooth germ mesenchyme cells; tooth regeneration.

Pituitary adenylate cyclase-activating peptide (PACAP)1-38 has been reported to be responsible for regulation of a disparate array of developmental processes in the central nervous system, and its antiapoptotic effect has been revealed in numerous models, pointing to its relevance in the etiology of neurodegenerative disorders. However, its function in retinal development remains unclear. Here, we aimed to point out that versatility can be achieved through interaction with other regulators, in which PACAP can act indirectly on the retinal microenvironment.

Wistar rats at age postnatal day 1 were injected intravitreally with PACAP or PAC1 receptor antagonist (PACAP6-38, M65) or VPAC1 antagonist (PG97-269) alone or in combination. Retinas were removed at 3, 6, 12, or 24 hours after injection. Changes in mRNA level were assessed using quantitative PCR, whereas changes in protein levels were measured by Western blot.

Intravitreal injection of PACAP or PAC1 receptor antagonists or the VPAC1 antagonist showed that PACAP receptors regulate the expression of five key secreted molecules (i.e., Fgf1, Bmp4, Wnt1, Gdf3, and Ihh), wherease other crucial morphogens (i.e., Fgf2, Fgf4, Fgf8, Fgf9, Shh, and Bmp9) were not affected. Pharmacologic dissection revealed that both PAC1 and VPAC1 induced downstream signaling and could cause upregulation of Fgf1, Bmp4, and Wnt1, whereas expression of Gdf3 might be mediated through the VPAC2 receptor.

Our data are the first to shed light on PACAP as a secretagogue regulating a sustained production of morphogens, which in turn could enable PACAP to serve as a mitogen for retinal cells, to induce ganglion cell differentiation, and to contribute to RPE development.

OBJECTIVE

The purpose of this study was to compare the effects of rhBMP2 with rhBMP9 on ridge augmentation following healing of extraction sockets in dogs.

METHODS

Five male Beagle dogs, approximately 12 months of age, were used. The mesial roots of the four maxillary premolars were endodontically treated. The distal roots were extracted, and the buccal bony walls removed. All extraction sockets were filled with deproteinized bovine bone mineral (DBBM). A collagen membrane was soaked with 4 μg or 20 μg of rhBMP9, 20 μg of rhBMP2 or sterile saline and placed over the augmented sites. All animals were euthanized after 8 weeks of healing and investigated by micro-CT and histologic analysis. A one-way ANOVA with Tukey's HSD post hoc test was used to compare the differences between the four groups.

RESULTS

New bone apposition in all defects was observed from the original bone. RhBMP samples showed an increase in bone formation in the buccal area and better integration of DBBM particles when compared to control sites. Both rhBMP9 defects showed higher values of bone (p = 0.024), bone marrow (p = 0.044), and total augmentation volume (p = 0.033) than the rhBMP2 (20 μg) or control sites. Highest bone area was found in rhBMP9 defects (p = 0.895).

CONCLUSIONS

Within the limitations of the present study, rhBMP9 sites demonstrated higher bone-inducing potential in combination with DBBM than rhBMP2. While rhBMP9s failed to demonstrate a clear dose-response relationship to the outcomes, future studies are necessary to evaluate the appropriate dose and carrier systems.